LncRNAs harbouring regulatory motifs within repeat elements modulate immune response towards COVID‐19 disease severity and clinical outcomes

To the Editor: Modulators of COVID-19 differential disease severity and clinical outcome deserve focused attention. In the background of paucity of comprehensive elucidation of the functional role of lncRNAs in COVID-19 clinical subphenotypes (mild, moderate, severe and mortality), albeit infected by the same pathogen SARS-CoV-2, we undertook this novel study in a hospital admitted cohort of 117 patients in India. Our integrative analysis highlights important role of lncRNAs in regulating immune response with plausible functional role of transcription factor binding sites (TFBS) within the repeat elements of the significant differentially expressed (DE) lncRNAs. Hospitalised COVID-19 patients were stratified into subphenotypes based on their distinct disease phenotypes and outcomes as per Indian Council of Medical Research (ICMR). Patients outside the ICMR guidelines were stratified into two groups: respiratory support (RS) and shortness of breath (SOB) (detailed methodology, results and literature supporting data interpretation as File S1). Figure 1A summarises study highlighting patient segregation into subphenotypes, experimental methodologies and downstream analysis for lncRNA differential expression, lncRNA–miRNA–mRNA interaction, pathway enrichment and role of regulatory sites with the repeat elements. The patient demographics and clinical data are summarised in File S1: Table S1, wherein, the median Ct value of E/RdRp gene was significantly different between recovered/mortality and RS/SOB patients, respectively (Figure 1B,C). SpO2 level was significantly different between recovered/mortality and mild/moderate/severe patients (Figure 1D), in addition tomedian age being different between mild/moderate/severe and mortality patients (Figure 1E). The duration of hospital stay was also significantly different in mild/moderate/severe and RS/SOB categories (Figure 1F).


LncRNAs harbouring regulatory motifs within repeat elements modulate immune response towards COVID-19 disease severity and clinical outcomes
To the Editor: Modulators of COVID-19 differential disease severity and clinical outcome deserve focused attention. In the background of paucity of comprehensive elucidation of the functional role of lncRNAs in COVID-19 clinical subphenotypes (mild, moderate, severe and mortality), albeit infected by the same pathogen -SARS-CoV-2, we undertook this novel study in a hospital admitted cohort of 117 patients in India. Our integrative analysis highlights important role of lncRNAs in regulating immune response with plausible functional role of transcription factor binding sites (TFBS) within the repeat elements of the significant differentially expressed (DE) lncRNAs.
Hospitalised COVID-19 patients were stratified into subphenotypes based on their distinct disease phenotypes and outcomes as per Indian Council of Medical Research (ICMR). Patients outside the ICMR guidelines were stratified into two groups: respiratory support (RS) and shortness of breath (SOB) (detailed methodology, results and literature supporting data interpretation as File S1). Figure 1A summarises study highlighting patient segregation into subphenotypes, experimental methodologies and downstream analysis for lncRNA differential expression, lncRNA-miRNA-mRNA interaction, pathway enrichment and role of regulatory sites with the repeat elements.
The patient demographics and clinical data are summarised in File S1: Table S1, wherein, the median C t value of E/RdRp gene was significantly different between recovered/mortality and RS/SOB patients, respectively ( Figure 1B,C). SpO 2 level was significantly different between recovered/mortality and mild/moderate/severe patients ( Figure 1D), in addition to median age being different between mild/moderate/severe and mortality patients ( Figure 1E). The duration of hospital stay was also significantly different in mild/moderate/severe and RS/SOB categories ( Figure 1F). To understand role of lncRNAs in modulating host response in the patients who succumbed to COVID-19, we performed differential expression analysis between recovered and mortality patients. We found three lncR-NAs significantly downregulated in the mortality patients (File S2; Figure 2A), with Figure 2B highlighting top 20 DE lncRNAs. Integration of lncRNA-miRNA-mRNA regulatory potential revealed that by virtue of LINC00174:11 downregulation in mortality, miR-1910-3p-mediated elevation of NF-kB signalling and cytokine storm were possible. 1 Downregulation of RNASEH1-AS1:23 and ROR1-AS1:6 may modulate heightened immune, inflammatory and stress response, as well as viral replication during mortality, mediated by miR-218-5p and miR-375. 2,3 DEG and GSEA analysis of study cohort in conjunction with LncRNA-miRNA-mRNA interaction network, highlight heightened inflammatory response (Files S3-S5; Figure 2C,D).
Subsequently, to elucidate the role of lncRNAs in modulating host response to COVID-19 disease trajectories, we identified DE lncRNAs among COVID-19 subphenotypes (mild vs. moderate/severe/mortality, moderate vs. severe/mortality and severe vs. mortality) ( Figure 3A,B; File S2). We observed LINC00294:1 upregulation and LINC00504:9 and RNASEH1-AS1:23 downregulationmediated decreased inflammatory responses in the moderate (vs. mild), whereas MALAT1 downregulationindicated heightened immune response in moderate patients. 4 The downregulation of UGDH-AS1:11 in the severe indicates a MOV10 and UPF1-mediated decreased antiviral response. 5 Downregulation of LINC00504:9 also indicates decreased immune response in the severe patients. In the mild versus mortality patients, we observed downregulation of MALAT1:9, LINC00504:9 and RNASEH1-AS1:23 in the mortality. Downregulation of LINC00504:9 and RNASEH1-AS1:23 suggests a decreased inflammatory and antiviral response in the mortality  patients, whereas downregulation of MALAT1:9 suggests an increased innate immune response in the mortality, contrary to other findings. 6 Upregulation of LUCAT1:3 in mortality indicates activation of interferon immunity, whereas downregulated LINC01537 reflects increased iNOS-mediated stress and decreased T-cell activation in mortality. 7,8 MALAT1:9 upregulation in the severe (vs. moderate) indicates decreased immune response in severe, whereas UGDH-AS1:11 downregulation suggests decreased antiviral response and increased disease severity in the severe. 5 Finally, LINC00273 downregulation in the mortality group (vs. severe) could possibly explain the decreased early innate immune response in mortality.

F I G U R E 2 Differentially expressed lncRNAs in the recovered patients (compared to mortality) and functional analysis. (A) Volcano plot
showing the differential expression of lncRNAs in recovered versus mortality patients. The green dots represent lncRNAs with more than log2 fold change of ±2 only, while red dots represent lncRNAs significant based on log2 fold change and q-value both. (B) Differential expression profile of top 20 lncRNAs (based on log2 fold change) based on average normalised counts between recovered and mortality patients. (C) lncRNA-miRNA-mRNA interaction network, brown box represents miRNA, purple circle represents lncRNAs and green circle represents mRNAs. (D) Gene set enrichment analysis of the genes interacting with the differentially expressed lncRNAs, where x-axis represents the normalised enrichment score (NES), and the colour represents the direction of NES For understanding the possible mechanism of gene regulation by specific lncRNAs, we analysed for repeat element distribution within the DE lncRNAs, with focus on LINE and SINE repeat elements (File S6). We observed significantly higher distribution of SINE/Alu, SINE/MIR and LINE/L1 elements across the comparison groups ( Figure 4A,B). The distribution of SINE/Alu and LINE/L1 was higher in our DE lncRNA compared to that of overall distribution of these repeats (File S6; Figure S3A,B). Importantly, higher presence of Alu elements (SINE) was found in the DE lncRNAs in the mortality patients. This indicates heightened stress response during mortality as highlighted by existing literature, suggesting functional role of Alu repeats during viral infection. 10 Subsequently, we analysed for the genes present within 5 kb upstream and downstream of the seven lncRNAs (12 genes) and performed pathway enrichment analysis to understand the biological functions of the genes (File S7; Figure 4C). Based on the pathway enrichment analysis, we selected TRPM2-AS1 and RNASEH1-AS1, and explored the role of TFBS in regulating the overlapping genes. We found four TFs (majorly bind to TFBS within Alu and L1 elements in TRPM2-AS1), SOX2, GATA3, FOXO1 and FOXO3, to regulate the TRPM2 gene expression, while TFs, SOX10 and GATA6, bind with RNASEH1-AS1 to regulate the RPS7 expression ( Figure 4D). This highlights possible TFBSmediated regulation of genes upstream/downstream of the lncRNAs.
In summary, our study highlights lncRNA-mediated dysregulation of immune and stress responses and their potential mechanism during the early phase of SARS-CoV-2 infection, which potentially modulates different degrees of disease severity subphenotypes: mild, moderate, severe and mortality.

A C K N O W L E D G E M E N T S
The authors duly acknowledge all the COVID-19 patients who participated in the study. Authors acknowledge the help and support from Dr. Aradhita Baral towards facilitation as research manager and coordination with the funders. Authors acknowledge the support of Anil Kumar and Nisha Rawat towards COVID-19 sample transport and sample management. Priti Devi, Partha Chattopadhyay, Kriti Khare and Aanchal Yadav acknowledge the CSIR for their research fellowship.

C O N F L I C T O F I N T E R E S T
The authors declare that there is no conflict of interest.